Somatostatin and Its Clinical Uses:
Somatostatin is a polypeptide having numerous physiological and pathological actions associated with the regulation of hormone, neurotransmitter and exocrine secretion as well as cell growth and differentiation. These actions of somatostatin are mediated by high affinity plasma membrane receptors in responsive cells.
Somatostatin receptors are present in many human cancers including pituitary adenomas, gastroenteropancreatic tumors and breast and prostate carcinomas. Hypersecretion of hormone by secretory tumors can be controlled by administration of a somatostatin analog which binds to and activates a somatostatin receptor. For example, when treated in vitro with the somatostatin analog octreotide, most receptor-expressing tumors respond with decreased hormone secretion and growth, however, some tumor cells react to this analog with stimulated growth. (Lamberts et.al., 1995, Ciba Foundation Symposium 190: Somatostatin and its Analogs p. 222-239.)
Somatostatin receptor-positive tumors may be visualized by administration of a labeled analog of somatostatin and determining the localization of the label. Discrepancies in binding studies using various somatostatin analogs originally demonstrated the presence of somatostatin receptor subtypes in tumors. Specific knowledge of the somatostatin receptor subtype present in a tumor is required to determine if a specific analog such as octreotide would be of therapeutic or diagnostic value in the treatment or localization of a specific patient's tumor.
Somatostatin Receptors:
Somatostatin interacts with specific membrane receptors to initiate a cellular response. Somatostatin receptors belong to a class of G-protein associated receptors having similar predicted three-dimensional structures consisting of seven transmembrane domains bridged by extracellular and intracellular loops. The predicted structure of the human sst2 receptor is shown in FIG. 1.
The somatostatin receptor family includes at least six distinct receptor subtypes encoded by five different genes, one of which generates two splice variant mRNAs. Gene sequences encoding human, rat, and, in some cases mouse somatostatin receptor (sst receptor) subtypes 1, 2, 2b, 3, 4 and 5 have been published in the literature (Bruns et al., Ann. NY Acad. Sci., 733:138-146, 1994 and references cited therein). FIG. 2 shows deduced amino acid sequences of the carboxy terminus of each known receptor subtype beginning at the carboxy terminal end of the seventh putative membrane-spanning domain and extending intracellularly. As shown, there is high homology between species within each receptor subtype, but little homology between receptor subtypes.
The basic and clinical study of somatostatin receptors as well as other seven transmembrane domain receptors has been hampered by the inability to generate specific, high affinity receptor antibodies. Such antibodies are essential for the rapid isolation of receptor proteins for biochemical studies, (e.g., by immunoprecipitation), and for efficient identification and quantification of receptors in tissues and cells, (e.g., by immunocytochemistry and Western blot methods).
There are many reports describing production of anti-peptide antibodies to different regions of G-protein coupled hormone receptors. These antibodies vary markedly in their ability to bind the receptor in its native or denatured form, with many having such low affinity for the receptor that they are not practically useful. No overall strategy for identifying a useful peptide antigen from a somatostatin receptor has been successful.
Antibodies to somatostatin binding proteins:
Purified receptor protein and fragments have been postulated as antigens to generate anti-somatostatin receptor antibodies. (European Patent Appln. EP92105164.5 to Eppler; U.S. Pat. No. 5,331,094.) However, to date no such antibody has been generated and shown to bind true G-protein coupled somatostatin receptors with high affinity and with the ability to discriminate between receptor subtypes.
Antibodies against somatostatin binding proteins which are unrelated to the seven transmembrane domain somatostatin receptor have been generated. (Theveniau et.al., J.Neurochem.63:447-455, 1994; LeRomancer et.al., J.Biological Chem. 269:17464-68, 1994; Reyl-Desmars et.al., J. Biological Chem. 264:18789-95, 1989; Theveniau et.al., PNAS 89:4314-4318, 1992; Nakabayashi et.al, Hybridoma 11:789-794, 1992.) In one report, a 90 kDa somatostatin binding protein was isolated from a gastric cell line (Reyl-Deymars et al., J. Biol. Chem. 264:18789-18795, 1989) using a monoclonal antibody prepared to a partially purified, membrane protein preparation. LeRomancer et al. subsequently cloned the 90 kDa protein antigen and showed that it was unrelated to the seven-transmembrane domain somatostatin receptor family. A functional role for this somatostatin binding protein in somatostatin signaling has not been demonstrated.
Theveniau et al, (Proc. Natl. Acad. Sci. 89:4314-4318 1992), described the production of polyclonal antibodies to a size-fractionated preparation of solubilized brain membrane proteins. The resulting antiserum was microfractionated by elution from immunoblots of size fractionated brain proteins. One of the antiserum fractions contained antibodies which could precipitate a somatostatin binding activity with low efficiency (&lt;2%) and also bound a 60 kDa protein on immunoblots. This putative "somatostatin receptor antibody" was later used by these investigators to localize a "receptor antigen." (Theveniau et al., J Neurochem. 60:1870-1875, 1993.) The recited antibodies do not bind any of the known seven-transmembrane domain somatostatin receptors, as shown in Theveniau et al, 1993. Moreover, the protein recognized by this antibody has never been isolated, cloned, or sequenced. Thus, any relationship of the protein recognized by this antibody to the known somatostatin receptor subtypes is unknown.
Nakabayashi et al described the production of monoclonal antibodies against a crude preparation of soluble rat brain membranes. The selected antibodies partially inhibited somatostatin binding to soluble brain proteins and also inhibited somatostatin binding to a 100 kDa protein in non-denaturing gels. Antibody binding specificity for somatostatin receptors, binding affinity, and ability to directly bind and/or discriminate between receptor subtypes was not disclosed. The paper provides no evidence that the antibodies actually bind directly to any of the known seven-transmembrane domain somatostatin receptor subtypes.
Antibodies to somatostatin receptors:
It has been proposed that antibodies could be raised against cloned sst receptors or expressed fragments thereof (e.g., Bell et al., WO 93/13130). However, to date, no such antibodies have been reported and shown to recognize and discriminate between somatostatin receptor subtypes. Several laboratories have generated antibodies to polypeptides having sequences derived from portions of the cloned somatostatin sst2 receptor. Patel et al (J. Biological Chem., 269:1506-1509, 1994.) generated antibodies against a nine amino acid peptide corresponding to the extracellular amino-terminal segment containing residues 35-42 of the human sst2 receptor. Although the resulting antibodies partially inhibited the binding of somatostatin to rat brain and pituitary cell membranes, they were not shown to immunoprecipitate any receptor protein. Further, the authors did not demonstrate any inhibition of binding that was specific for the sst2 receptor subtype. In Western blots, the antibody of Patel et al. recognized a protein of approximately 72 kDa. However, the authors did not show immunostaining specific for the sst2 subtype. Moreover, the intensity of the immunostaining was not shown to be proportional to receptor density, in fact, the weakest staining was observed in sst2 receptor-expressing COS cells which should have contained the highest receptor concentration of the cells tested. Therefore, the reference does not teach or suggest any specificity of this antiserum for different sst receptor subtypes.
Theveniau et al., (J. of Neurochemistry 63:447-455, 1994) generated antibodies against two different peptides with sequences derived from the sst2 receptor, one from the predicted third extracellular loop (2e3) and the other from the predicted C-terminal cytoplasmic tail of the receptor (2i4) (See FIG. 1). The 2e3 antibody did not specifically immunoprecipitate the ligand-receptor complex from CHO cells expressing the sst2 receptor. The 2e3 antibody was shown to detect a 148 kDa protein in rat brain, rat pituitary, and rat Ar4-2J cells. However, the somatostatin receptors normally found in these cells and tissues have previously been shown to have substantially lower molecular weights by photoaffinity labeling. The apparent molecular weight of Ar4-2J cell somatostatin receptors is 75-80 kDa (Brown, P. J., J. Biol. Chem. 265:17995-18004, 1990; Viguerie, N., Am. J. Physiol. 255:G113-G120, 1988). The molecular weight of rat pituitary somatostatin receptors varies between 82 kDa and 94 kDa (Kimura, J. of Biol. Chem. 264:7033-7040, 1989). The molecular weight of rat brain somatostatin receptors is approximately 71 kDa (Kimura et al., Biochem. Biophys. Res. Commun. 160:72-78, 1989). Moreover, somatostatin receptors normally migrate as broad bands on SDS polyacrylamide gels, as has been observed for other glycosylated seven-transmembrane domain receptors (above references). In contrast, the 148 kDa band recognized by the 2e3 antiserum was very sharp. Both because of its uncharacteristic molecular weight and because of its unusual migration pattern on SDS-PAGE, the 148 kDa protein detected by the 2e3 antibody is unlikely to represent the sst2 receptor.
The 2i4 antibody of Theveniau et al. immunoprecipitated 10-15% of solubilized sst2 receptors prepared from CHO cells expressing recombinant sst2 receptors, but was unable to detect any specific protein in immunoblots. The affinity and specificity of this antibody was not disclosed in the reference, however, very high serum concentrations (20 .mu.l) were used to achieve the modest immunoprecipitation reported. In studies that will be described below, antibodies were prepared against a larger peptide antigen which included the Theveniau 2i4 peptide. This larger peptide antigen was expected to have greater antigenicity than the 2i4 peptide and to produce antibodies of higher specificity for the sst2 receptor. However, this larger peptide was unable to induce antibodies having high affinity and specificity for the sst2 receptor (see Example 2).
In European patent application EP 92105164.5, Eppler purports to have raised an antibody against an eight amino acid peptide from the carboxy terminus of the sst2 receptor. Antisera from immunized animals was shown to detect purified sst2 receptor protein on a Western blot. However, the antiserum also bound to the enzyme, Endo F, which was used to deglycosylate the receptor protein and which was the only other protein in the sample. Therefore, the antibody cannot be said to have specificity for sst receptors. Moreover, there was no indication that the antibody had the sensitivity necessary to detect sst receptors in a heterogenous, unpurified preparation.
Cloned Somatostatin Receptors:
Bell et al, WO 93/13130, discloses the gene sequence encoding seven transmembrane domain somatostatin receptors sst1 and sst2 and recites methods for producing antibodies to the cloned receptors using recombinant proteins or protein fragments derived from the DNA sequence. This reference fails to identify any particular sequence of the cloned sst1 or sst2 receptor which would successfully induce antibodies, and importantly, does not disclose or suggest any sequence of the cloned receptors as inducing antibodies having the ability to recognize a specific receptor subtype.
Somatostatin receptor subtypes:
The ability to discriminate between somatostatin receptor subtypes in a sample tissue is critical to the development and use of somatostatin analogs in diagnostic and therapeutic methods targeting a specific receptor subtype. For example, the somatostatin analog OCTREOTIDE.RTM. (also known as SANDOSTATIN.RTM. or SMS 201-995), interacts with the sst2, and sst5 receptors, and to some extent with the sst3 receptor, but not with sst1 and sst4 receptors. This analog has been approved for clinical use in the treatment of certain tumors which express sensitive sst receptors. (Pless, J. Digestion, 54 (Suppl. 1):7-8, 1993) To date there is no quick, easy, and precise method to determine if a tissue or tumor sample expresses a receptor subtype which can be effectively treated by an analog such as SANDOSTATIN or any other receptor subtype-specific somatostatin analog.
It would be very useful to provide antibodies for efficient and predictable screening of sample tissues to identify those expressing specific somatostatin receptor subtypes. Such antibodies must have a high affinity and specificity for a particular receptor subtype and be capable of specifically recognizing the seven transmembrane domain somatostatin receptor subtype by either immunocytochemical staining, immunoblotting, immunoprecipitation, on an ELISA-type assay.